Ph.D. Thesis: “Protective immune response in P.falciparum ...shodhganga.inflibnet.ac.in/bitstream/10603/9014/10/10_chapter 1.pdfwith protection from clinical malaria30. MSP-1

Ph.D. Thesis: “Protective immune response in P.falciparum malaria” 2011

S.D. Lourembam 16

CHAPTER I:

Introduction


S.D. Lourembam 17

1. INTRODUCTION

Malaria remains a major global health problem with 300 to 500 million

clinical infections and more than a million deaths reported each year. In 2009,

World Health Organization (WHO) reported 225 million malaria cases and

781000 deaths from 106 endemic countries. Majority of the cases were from

African countries (78%) and the contribution from South East Asian and

Mediterranean regions were 15% and 5% respectively. About 85% of the deaths

caused by malaria were in children under 5 years of age1. It is commonly

associated with poverty and is also an important cause of poverty slowing down

economic growth by 1.3% per year in endemic areas. A 10% reduction in disease

was associated with 0.3% higher growth2. In India, retrospective analysis of

burden of malaria showed that disability adjusted life years lost due to it was

1.86 million years3.

In the South East Asia Region (SEAR), approximately 70% of the total

population is at risk of malaria, of which three countries, India (65%), Myanmar

(20%) and Indonesia (12%) accounted for 94% of the reported cases1. 60.5% of

all infection were caused by Plasmodium falciparum. Malaria is endemic in the

Indian state of Assam contributing more than 5% of the total cases recorded in

the country annually. Malarial outbreaks characterized by enhanced morbidity

and mortality are common across the state4. The endemicity of malaria is not

uniform across the state with many pockets along forest fringes, forest and

foothill villages particularly along the inter-country/inter-state border vulnerable

to malaria outbreaks5. These high risk zones are predominantly inhabited by

indigenous tribal populations of distinct ethnic backgrounds. Most of these

affected areas report widespread resistance to first line drugs like chloroquine,

sulphodoxine-pyrimethamine and even quinine resistance is being reported. In

fact, resistance to chloroquine was first reported from Karbi Anglong district of

Assam in India6.

Human malaria is caused by four distinct species of Plasmodium viz P.

falciparum, P. ovale, P. malariae and P. vivax. The malarial parasite has a


S.D. Lourembam 18

complex life cycle (Fig 1.1) involving both female anopheline mosquito (sexual

phase) and human (asexual phase) as host.

Fig 1.1: Life cycle of Plasmodium falciparum. (Adapted from Crompton et al.

2010).

Malaria causes a wide variety of symptoms ranging from asymptomatic

infection to very mild symptoms to severe (complicated) disease and even death.

Based on the clinical presentation, the disease may be divided into

uncomplicated and complicated malaria as per WHO guidelines7.

Strategies to control malaria have been redefined with effective diagnosis

and treatment measures along with vector control interventions like Insecticide


S.D. Lourembam 19

Residual spray and use of insecticide treated mosquito nets. However, with

emergence of insecticide resistant mosquitoes and the parasites developing

resistance against conventional antimalarial drugs, it poses challenge to malaria

control. Recently, artemisinin resistance has been reported from the Greater

Mekong subregion and the threat of its spread to other areas is a serious issue1.

Notably, chloroquine resistance in P. falciparum was first detected from South

East Asia region8.

Vaccination is considered an important control measure which has long

been pursued but with little progress. Studies of various vaccine candidates

including those of the liver stage and asexual blood stage candidate reported

limited success in earlier attempts9. Combination B vaccine consisting of

merozoite surface proteins 1 and 2 (MSP-1, MSP-2) and ring stage infected

erythrocyte surface antigen (RESA) showed a 62% reduction in parasite density

in vaccinees10. Recently a malaria vaccine candidate RTS, S, has entered phase

III trial in Kenya11. The difficulties in development of a malaria vaccine may be

attributed to the complex life cycles and antigenic polymorphism exhibited by

the parasite and compounded by a poor understanding of protective immune

mechanism12. Besides, the disease shows variation in epidemiology across

different endemic regions13.

The genetic structure of P. falciparum has been demonstrated to be

highly diverse world over14. Major vaccine candidate genes exhibit antigenic

diversity in the form of allelic polymorphism where the alternate forms of

antigen coding genes exist. Mention can be made of the merozoite surface

proteins (MSPs), the serine repeat antigen (SERA) genes families. Additionally

diversity may be generated by clone multiplicity where a clonal lineage of

parasite expresses alternate forms of an antigen without changes in genotype.

Variations in genes encoding antigens are also generated by non meiotic

recombination such as strand slippage events, gene conversion as well as

homologous recombination during meiosis16. This extensive diversity in

Plasmodium falciparum antigens is seen as immune evasion mechanism of the

parasite. Repetitive sequences constitute immunodominant epitopes in parasite


S.D. Lourembam 20

proteins, therefore sequence variation in allelic forms of the molecules may

prevent the population from developing immunity to different strains of the

parasite17. Elucidating the extent of genetic variation in the malaria parasite will

therefore be central to decreasing the malaria disease burden18. The non uniform

population structure of P. falciparum worldwide depends upon local factors

related to parasite, vector and host factors19. Factors like transmission intensity

and drug treatment too influence the genetic diversity of the parasite14.

Allelic diversity and size polymorphism of merozoite surface protein

1(MSP-1) gene have been employed as a molecular marker in studies of malaria

transmission dynamics and host immunity in P. falciparum malaria. MSP-1 gene

has also been extensively studied as it has a role in invasion, disease and immune

evasion14. MSP-1 gene is divided into 17 blocks, based on analysis of sequence

diversity. Block 2 region near the N terminus and block 17 at the C terminus are

major targets of host immunity. The block 2 of MSP-1 is described by 3 distinct

allelic families MAD20, K1 and RO33 and contains antigenically unique

tripeptide repeats with extensive diversity in the number of repeats20.

Significantly, block 2 repeats are targeted by P. falciparum-inhibitory

monoclonal antibodies and naturally acquired antibodies associated with clinical

immunity in humans15. A comprehensive understanding of the immune responses

lies in dissecting the antigenic diversity of P. falciparum.

Immune response to malaria is complex and is species and stage specific.

Immunity to malaria can be either anti disease immunity which confers

protection against clinical disease or anti infection immunity conferring

protection against parasitaemia21. Naturally acquired immunity to malaria

develops slowly and is rarely sterile. In holoendemic or hyperendemic areas

older population generally develops immunity showing asymptomatic

parasitaemia while in areas of low malaria transmission, individuals do not

develop immunity in an age dependent manner. The infection and disease can

therefore occur in both children and adults22. The picture that emerges from

human studies is that immunity to malaria infection is relatively slow to develop


S.D. Lourembam 21

and incomplete, although immunity to disease is acquired more quickly and may

be important after a single episode23. Acquired immunity involves both the

antibody mediated and cell mediated immune response.

Malaria infection induces strong humoral immune responses, involving

production of predominantly IgM and IgG but also other immunoglobulin

isotypes. Species as well as stage specific antibodies against wide variety of

parasite antigens have been reported while a large proportion of the antibodies

are non-malaria specific24. The importance of antibodies as mediators of

protective immunity to malaria is well established in both animal and human

infections. Mice lacking B cells were unable to clear parasites from P. chabaudi

chabaudi AS infection, rather such mice developed chronic parasitaemia25.

Passive transfer of monoclonal antibodies against parasite antigens conferred

protection in naive mice by reducing parasitaemia and clinical disease 26. In

humans, treatment of P. falciparum-infected patients from Thailand with IgG

extracted from African immune adults, resulted in reduction of parasitemia and

clinical symptoms27. Studies in humans and mice show that memory B cells are

either poorly induced or short lived as a result of infection. Recent studies

showed that memory B cells and antibodies increased gradually over many years

and were dependent on cumulative exposure to malaria9. Despite the importance

of antibody responses for protection against malaria, not all antibodies are

reported to be protective. Polyclonal antibody specific to MSP-2 but not

monoclonal specific to the same antigen enhanced invasion of multiple

merozoites into RBC28.

Protective antibodies primarily target the asexual stage antigens of which

MSPs are leading blood stage vaccine candidate molecules of Plasmodium

falciparum. MSP-1 is the major surface protein of the parasite, which is

synthesized as a 190 kDa precursor protein16. It undergoes proteolytical cleavage

into four fragments and remains attached to merozoite surface by

glycoslyphosphatidylinositol anchor prior to invasion. During erythrocyte

invasion, majority of the MSP-1 complex is shed of which only the 19 kDa C-

terminal fragment remains anchored on the merozoite surface29. Antibodies


S.D. Lourembam 22

targeted to this fragment are shown to inhibit erythrocyte invasion and associated

with protection from clinical malaria30. MSP-119 fragment has a highly

conserved sequence however at least six single nucleotide polymorphisms

(SNPs) have been identified in its two epidermal growth factor like (EGF-like)

domains31. Allele specific as well as cross-reactive antibody responses to variants

of MSP-119 fragment have been reported32. Further, certain MSP-119

polymorphisms have been implicated as particularly important to immunity31.

Cell mediated immunity (CMI) has crucial roles in protective immunity

to malaria but also has the potential to cause tissue pathology and contribute to

the development of severe malaria33. Vaccine formulations with T cell epitopes

induce a strong memory response9. The CD4 T-cell subset is of major

importance for the induction of blood-stage immunity in both murine and human

malaria, while the CD8 subset has been shown to be cytolytic against liver stages

of the parasite28. In experimental mice CD4 T cells could act independent of B

cells in resolution of parasitaemia34. Humans lacking previous exposure to P.

falciparum as well as malaria exposed individuals have CD4 T cells that

proliferate and secrete IFNγ in response to parasite antigen and inhibit parasite

growth in vitro35. Another subset of CD4+ T lymphocytes, T-regulatory (T-reg)

cells is postulated to be involved in immunosuppression where effectors

responses were enhanced in the absence of Tregs33.

T cells also play a central role in the elimination of blood stage malaria

parasites through the release of cytokines that activate other effectors cells28.

Specific cytokine profiles are associated with different clinical manifestations.

Th1 cells activated macrophages and other cells to produce mediators through

release of inflammatory cytokine36. Th1 were seen to be responsible in the initial

resolution of acute parasitemia through production of IFNγ while Th2 were

required for eventual clearance of the parasites via T-B cell cooperation37. The

balance between the Th1 and Th2 immune response may determine the level of

parasitaemia and disease outcome. An early and sustained Th1 response was

critically linked to IL-12 through IFN-γ production38. Recent evidence suggest

that rapid cell mediated immune responses are contributed by effector cells of


S.D. Lourembam 23

NK and T cells lineages which contain the initial stages of malaria through IFN-γ

production39.

Natural killer (NK) cells are lymphocytes of the innate immune system

that are involved in the early defense against foreign cells and autologous cells

undergoing various forms of stress, such as microbial infection or tumor

transformation. NK cells also influence adaptive immunity by modulating

dendritic cell function and by inducing Th1 polarization via IFN-γ production40.

NK cells and NK Receptor (NKR) positive cells are suggested to significantly

control susceptibility and resistance to both malaria infection and severe disease

syndromes. This was seen to depend on the receptors encoded within the Natural

Killer Cell Receptor Complex (NKC) 41. NK cells were seen to have direct

contact with P. falciparum infected red blood cell via NK Cell receptors42.

Further, NKR positive γδT cells were seen as the major source of IFN γ in

response to P. falciparum infection in humans as well as in murine models which

were in part controlled by NKR loci41.

NKRs are largely encoded by two genetic loci in humans: the Natural

Killer Complex (NKC) on chromosome 12 and the Killer cell Immunoglobulin-

like receptors (KIRs) region on chromosome 19. Diverse set of inhibitory and

activating NKRs controlled NK cell activation and the outcome of NK cell

activity is the balance of signals from activating and inhibitory receptors43.

The KIR gene complex is characterized by variation in gene content.

Studies suggest that they are rapidly evolving genes which lack conservation

among species and exhibit remarkable diversity as haplotypic variation in gene

number and content and allelic polymorphism of individual genes43, 44. Two

broad haplotype groups termed A and B are used to define variation in KIR

genetic profile44. Frequencies of specific KIR haplotypes vary across different

ethnic populations. The important role of KIR in the immune response and its

genomic diversity coupled with its specificity for HLA ligands, affects resistance

and susceptibility to pathogenesis of a number of infectious and autoimmune

diseases43. NK cells response to parasitized RBCs was noted to vary significantly


S.D. Lourembam 24

between individuals and that the variation was associated with KIR genotype of

an individual42.

North East India is inhabited by people of various ethnic origins with a

history of malaria endemicity. Malaria remains a major public health problem in

the state of Assam. Nearly 65% of the total population of the state (26.6 million)

is estimated to be living in high-risk areas45. Malaria transmission is perennial

and continues to be uninterrupted supported by major vectors namely An.

minimus, An. fluviatilis and An. dirus. The region is highly receptive to malaria

transmission due to excessive and prolonged rainfall (2–3 m) promoting vector

breeding and longevity due to high humidity (60–90%) and warmer climates

(22–33C) for most of the year46. P. falciparum is the major malarial infection

and accounts for 58−68% of the cases and the remainder are due to P. vivax.

Despite efforts to contain the disease mortality and morbidity are significantly

high due to emergence of drug resistant Pf malaria, delayed diagnosis due to

non-availability of facilities in interior villages, hilly areas and tribal belts4.

Malaria in Assam is poorly investigated with respect to diversity of the

circulating local Plasmodium falciparum and immune responses of the

population. Besides it is populated by individuals of different ethnic origin. The

present study was thus undertaken to understand the diversity of the Plasmodium

falciparum of the region and the host parasite interactions with an aim to

evaluate the protective immune mechanism and markers of protection if any.


S.D. Lourembam 25

Objectives:

1) To study the clone multiplicity of the parasite genotypes existing in

the study area

2) To elucidate the protective humoral immune response.

3) To elucidate the protective cell mediated immune response

Documents

Ph.D. Thesis: “Protective immune response in P.falciparum ...shodhganga.inflibnet.ac.in/bitstream/10603/9014/10/10_chapter 1.pdfwith protection from clinical malaria30. MSP-1